Longer Forms of Amyloid β Protein: Implications for the Mechanism of Intramembrane Cleavage by γ-Secretase

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Abstract
γ-Cleavage of β-amyloid precursor protein (APP) in the middle of the cell membrane generates amyloid β protein (Aβ), and ϵ-cleavage, ∼10 residues downstream of the γ-cleavage site, releases the APP intracellular domain (AICD). A significant link between generation of Aβ and AICD and failure to detect AICD41-99 led us to hypothesize that ϵ-cleavage generates longer Aβs, which are then processed to Aβ40/42. Using newly developed gel systems and an N-end-specific monoclonal antibody, we have identified the longer Aβs (Aβ1-43, Aβ1-45, Aβ1-46, and Aβ1-48) within the cells and in brain tissues. The production of these longer Aβs as well as Aβ40/42 is presenilin dependent and is suppressed by {1S-benzyl-4R-[1S-carbamoyl-2-phenylethylcarbamoyl-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamic acid tert-butyl ester, a transition state analog inhibitor for aspartyl protease. In contrast,N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a potent dipeptide γ-secretase inhibitor, builds up Aβ1-43 and Aβ1-46 intracellularly, which was also confirmed by mass spectrometry. Notably, suppression of Aβ40 appeared to lead to an increase in Aβ43, which in turn brings an increase in Aβ46, in a dose-dependent manner. We therefore propose an α-helical model in which longer Aβ species generated by ϵ-cleavage is cleaved at every three residues in its carboxyl portion.